Abstract: The wealth of information being obtained from genomic, proteomic, and glycomic research is allowing researchers to unravel the intricate genetic and epigenetic mechanisms associated with human health and disease. The intracellular delivery of nucleic acids to study these processes offers unprecedented promise for revolutionizing biomedical research and drug development. However, the nucleic acid delivery vehicle plays a central yet elusive role in dictating the efficacy, safety, mechanisms, and kinetics of gene regulation in a spatial and temporal manner;thus, having a far-reaching impact in health-related research. To this end, we have developed several novel carbohydrate-containing polymers that have shown outstanding affinity to encapsulate polynucleotides into nanoparticles (polyplexes) and facilitate highly efficient intracellular delivery without toxicity. The goals of this project directly commence from our previous work where we aim to examine our wide-range of delivery vehicles for their mechanistic pathways and kinetics of nucleic acid encapsulation and intracellular transport from the cell surface to their final intracellular destination. We plan to examine 10 different carbohydrate-based polymers synthesized in our laboratory for their delivery mechanisms and kinetics with three polynucleotide forms: plasmid DNA, oligodeoxynucleotide decoys, and small interfering RNA, in two cell types, H9C2(2-1) and Hela cells. The research program highlighted herein is driven by three specific goals: 1) to unravel the molecular-level interactions between structurally diverse yet analogous polymeric delivery vehicles and differing nucleic acid types and to correlate these interactions with the biological stability and mechanisms of the subsequent polyplexes, 2) to understand the interactions of these various polyplex types with cell surface glycosaminoglycans and compare polyplex structure to receptor selectivity and mechanisms of cellular uptake in two cell types. 3) To decipher the intracellular trafficking pathways in a spatial and temporal manner from uptake to the final destination for each polyplex form with the two model cell types. Public Health Relevance: The intracellular delivery of nucleic acids to study genetic and epigenetic processes associated with human health and disease offers unprecedented promise for revolutionizing biomedical research and drug development. However, it has been found that the nucleic acid delivery vehicle plays a central yet elusive role in dictating the efficacy, safety, mechanisms, and kinetics of gene regulation in a spatial and temporal manner. The goal of this project is to examine a wide range of novel nonviral glycopolymer-based delivery vehicles for their mechanistic pathways and kinetics of nucleic acid encapsulation and intracellular transport from the cell surface to their final intracellular destination with two models (cardiomyocytes and cancer cells) that represent the leading causes of disease and death.